Characterization and Localization of Digitoxin 12 ß-Hydroxylase from Cell Cultures of Digitalis lanata EHRH

Pharmazeutisches Institut der Universität, Auf der Morgenstelle 8, D-7400 Tübingen, Bundesrepublik Deutschland Z. Naturforsch. 43c, 199-206 (1988); received December 8, 1987 Cardiac Glycosides, Cytochrome P-450, Digitalis lanata, Digitoxin 12ß-Hydroxylase, Endoplasmic Reticulum The cytochrome P-450-dependent monooxygenase digitoxin 12ß-hydroxylase from cell cultures of Digitalis lanata needs NADPH and molecular oxygen and hydroxylates cardiac glycosides with the aglycon of digitoxigenin to the corresponding derivatives of the C-series. Other electron donors cannot replace NADPH. The apparent K"m-values are 26 UM for NADPH, 7.1 ÎM for ß-methyldigitoxin and 10 ÎM for digitoxin. The reaction is inhibited by NADP and cytochrome c in a competitive mode. The optimum temperature was at 20 °C. Low concentrations of Mn, Mg, and EDTA were slightly stimulatory, but there was no strict dependence on divalent cations. Digitoxin 12ß-hydroxylase is very stable at room temperature and the reaction proceeds for more than 20 h. After the addition of 15% glycerol, 70% of the original activity can be retained subsequent to freezing at —18 °C. By means of linear sucrose gradient fractionation of cellular membranes the digitoxin 12ß-hydroxylase was found to be located in the endoplasmic reticulum.